Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.
Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.
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DOI：
10.1021/bi401711n
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发表时间：
2014-04
期刊：
影响因子：
2.9
通讯作者：
Longmei Zhang;Huijuan Xu;Yanling Jie;Chao Gao;Wanjuan Chen;Shikui Yin;D. Samways;Zhiyuan Li
中科院分区：
文献类型：
--
作者：
Longmei Zhang;Huijuan Xu;Yanling Jie;Chao Gao;Wanjuan Chen;Shikui Yin;D. Samways;Zhiyuan Li
P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.
